Injection filler as well as preparation method and application thereof

Abstract

The invention discloses an injection filler and a preparation method and application thereof, the filler is polycaprolactone particles, the polycaprolactone particles are spherical particles, the particle size of the spherical particles is 20-100 [mu] m, and the polycaprolactone particles have rough surfaces or unsmooth surfaces. The polycaprolactone microspheres have rough surfaces or unsmooth surfaces, so that the contact area with cells is increased, the adhesion ability and residence time of the cells are improved, the affinity of the polycaprolactone microspheres is improved, and the growth of collagen can be stimulated and accelerated. In the preparation process, the dosage of each component is scientifically screened, and on the basis of controlling the viscosity of a water-phase solution, the spherical polycaprolactone particles which are regular, rough in surface and uniform in particle size are obtained in cooperation with a rapid membrane emulsification method.

Description

[0001] This case is a divisional application. The application number of the parent case is 202311046088.6, the application date is 2023.08.18, and the name of the invention is “An injectable filler, its preparation method and application”. Technical Field

[0002] The present invention relates to the technical field of soft tissue filling and medical cosmetic fillers, and in particular to an injectable filler and a preparation method and application thereof. Background Art

[0003] The skin is mainly composed of the epidermis and dermis, in which collagen exists in the dermis. With the physiological aging of people and various external stimuli, the loss rate of collagen in the dermis begins to accelerate, and the supply cannot keep up with the consumption, causing the skin to become loose and wrinkles to appear. Injection beauty is to inject fillers into the dermis or subcutaneously to improve the skin condition.

[0004] Biodegradable polymer particles have the property of decomposing in the body. Taking advantage of this property, they are widely used in implants such as plastic surgery fillers. At present, there are two main categories of these injection materials. One is the filling products represented by cross-linked sodium hyaluronate gel and collagen gel. The main mechanism of action is subcutaneous filling. After injection, the subcutaneous defects are expanded and filled, and wrinkles are smoothed. The other is the collagen stimulation products represented by polyesters such as poly-L-lactic acid (PLLA) and polycaprolactone microspheres. Polyester materials have the advantages of good biocompatibility, anticoagulant properties, non-toxicity and low immunity. They can be hydrolyzed and degraded into low molecular weight fragments in a physiological environment. The low molecular weight fragments can be endocytosed by macrophages and degraded in the cells. During the degradation process, by stimulating the host's immune response, a large number of subcutaneous macrophages and other immune cells are attracted, resulting in the proliferation of fibroblasts and the differentiation of myofibroblasts, synthesizing a large amount of collagen, thereby restoring subcutaneous capacity and repairing wrinkles.

[0005] At present, there are many products containing polylactic acid and / or polycaprolactone used to fill skin wrinkles and depressions, and their main forms are polylactic acid microparticles, polylactic acid microspheres and polycaprolactone microspheres. However, the hydrophilicity of existing medical polyester materials is generally poor, which is not conducive to the attachment of body proteins on their surface, thereby affecting the cell adhesion performance. In addition, medical polyester materials are prone to sedimentation in water-based solutions, which requires high user operation skills. In addition, the heterogeneity of the particle size will lead to phagocytosis by macrophages, decomposition before collagen is generated, or clogging of the injection needle.

[0006] CN104001209 and CN101842084 disclose PCL microparticles and their uses. Methylcellulose is used as a surfactant in the preparation method of microspheres. The microspheres have good flow properties, but the particle size distribution range of the microspheres is wide, and the size of the polymer cannot be well controlled. During use, the uneven particle size will affect the efficiency of cell phagocytosis, reduce the targeting of the particles, and easily cause adverse reactions such as needle blockage and bulging. In addition, since the surface of polycaprolactone microspheres is smooth and the adhesion ability to cells is poor, the effect is slow after injection into the subcutaneous layer, and the user experience is not good. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides an injectable filler and a preparation method and application thereof. The filler has good cell adhesion ability, uniform particle size, better dispersion stability, is not easy to block the needle, and has good biocompatibility and degradability.

[0008] One of the purposes of the present invention is to provide an injectable filler, wherein the filler is polycaprolactone particles, the polycaprolactone particles are spherical particles, the molecular weight of the polycaprolactone is 5000-30000, the particle size of the spherical particles is 20-100 μm, and the polycaprolactone particles have a rough surface or a non-smooth surface.

[0009] In a preferred technical solution of the present invention, the rough surface or non-smooth surface of the polycaprolactone particles has irregular pore sizes.

[0010] In a preferred technical solution of the present invention, the particle size of the spherical particles is preferably 20-70 μm, more preferably 25-50 μm.

[0011] In the preferred technical solution of the present invention, the molecular weight of the polycaprolactone is 10000-25000.

[0012] In the preferred technical solution of the present invention, the molecular weight of the polycaprolactone is 15000-22000.

[0013] Another object of the present invention is to provide a method for preparing an injection filler, comprising the following steps:

[0014] (1) Preparation of aqueous phase: Add surfactant into purified water, stir and dissolve;

[0015] (2) Preparation of organic phase: Add the injectable filler into the organic solvent and stir until completely dissolved;

[0016] (3) Emulsification granulation: The organic phase and the aqueous phase are mixed evenly, stirred, and emulsified using a membrane emulsifier to prepare microspheres;

[0017] (4) Stirring and volatilizing: Stirring to volatilize the organic solvent;

[0018] (5) Microsphere collection: Collect the suspension, separate, wash, and dry to obtain injectable filler microspheres.

[0019] In the preferred technical solution of the present invention, the surfactant is any one of polyvinyl alcohol, methyl cellulose, polyoxyethylene, polyoxypropylene, polyethyleneimine, polystyrene, polyoxysilane, hydroxypropyl cellulose or a combination thereof.

[0020] In the preferred technical solution of the present invention, the organic solvent is any one of acetone, ethanol, ethyl acetate, chloroform, and dichloromethane, or a combination thereof.

[0021] In the preferred technical solution of the present invention, the ratio of the surfactant to purified water is 10-50 g / L, preferably 15-40 g / L, and more preferably 20-30 g / L.

[0022] In a preferred technical solution of the present invention, the viscosity of the aqueous solution is 2-10 cP, preferably 4-8 cP.

[0023] In the preferred technical scheme of the present invention, during the preparation of the aqueous phase solution, purified water is added to the reaction kettle, mechanical stirring is turned on, and the rotation power is adjusted to 10-50 Hz, preferably 20-30 Hz; the surfactant is slowly added to the purified water, heated to 60-120° C., stirred for 2 to 6 hours, and cooled to room temperature after the surfactant is completely dissolved.

[0024] In a preferred technical solution of the present invention, the ratio of the injectable filler to the organic solvent is 100-300 g / L, preferably 150-250 g / L.

[0025] In a preferred technical solution of the present invention, during the preparation of the organic phase, the injectable filler is added to the organic solvent and stirred to completely dissolve it.

[0026] In the preferred technical scheme of the present invention, in the emulsification step, the mechanical stirring power is adjusted to 10-50 Hz, the organic phase is slowly added to the aqueous phase, and after stirring for 3-10 minutes, the pressure is adjusted to 0.5-1 bar using a membrane emulsifier, and a 10-50 μm membrane tube is used for rapid membrane emulsification.

[0027] In the preferred technical scheme of the present invention, in the stirring and volatilization step, the emulsified sample is transferred to a reaction kettle, the mechanical stirring power is maintained at 10 to 50 Hz, nitrogen / compressed air is used to replace the gas in the kettle, a flow rate of 1 to 20 m / s is maintained, and stirring is performed for 12 to 48 hours to completely volatilize the organic solvent.

[0028] In a preferred technical solution of the present invention, in the microsphere collection step, the microspheres are collected by centrifugation, sedimentation, filtration or a combination thereof.

[0029] In the preferred technical solution of the present invention, in the microsphere collection step, after the organic solvent evaporates, the suspension in the reaction kettle is collected, and the solid microspheres are separated using a centrifuge, the centrifuge speed is 2000-5000rpm, the centrifugation time is 2-8min, preferably the centrifuge speed is 3000-4000rpm, and the centrifugation time is 3-6min. In the preferred technical solution of the present invention, in the microsphere collection step, the collected microspheres are washed with purified water and ethanol, collected after centrifugation, and dried.

[0030] In the preferred technical solution of the present invention, the drying is carried out at 30-60°C for 10-30 hours, preferably at 30-40°C for 15-24 hours.

[0031] In the preferred technical solution of the present invention, the drying is air blast drying.

[0032] In the preferred technical solution of the present invention, in the cleaning step, purified water is used for cleaning 2-5 times, and ethanol is used for cleaning 1-2 times.

[0033] In the preferred technical solution of the present invention, the injectable filler is polycaprolactone microspheres.

[0034] In a preferred technical solution of the present invention, the polycaprolactone microspheres prepared by the method have a particle size of 20-100 μm and a rough surface or a non-smooth surface.

[0035] In a preferred technical solution of the present invention, the rough surface or non-smooth surface of the polycaprolactone particles has irregular pore sizes.

[0036] In a preferred technical solution of the present invention, the particle size of the spherical particles is preferably 20-70 μm, more preferably 25-50 μm.

[0037] In the preferred technical solution of the present invention, the molecular weight of the polycaprolactone is 5000-30000.

[0038] In the preferred technical solution of the present invention, the molecular weight of the polycaprolactone is 1000-25000.

[0039] In the preferred technical solution of the present invention, the molecular weight of the polycaprolactone is 15000-22000.

[0040] Another object of the present invention is to provide a preparation of an injectable filler, wherein the filler is polycaprolactone particles, the polycaprolactone particles are spherical particles, the molecular weight of the polycaprolactone is 5000-30000, the particle size of the spherical particles is 20-100 μm, and the polycaprolactone particles have a rough surface or a non-smooth surface.

[0041] In a preferred technical solution of the present invention, the preparation further comprises any one or a combination of a suspension stabilizer and a buffer.

[0042] In the preferred technical solution of the present invention, the suspension stabilizer is selected from any one of polyvinyl alcohol (PVA), gelatin, gum arabic, guar gum, chondroitin sulfate, hyaluronic acid (HA), cross-linked hyaluronic acid, sodium carboxymethylcellulose (CMC), polyvinyl pyrrolidone (PVP), methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), starch, pectic acid, heparin, glucose, β-cyclodextrin, chitosan, sodium alginate or a combination thereof. Preferably, the suspension stabilizer is hyaluronic acid, cross-linked hyaluronic acid and / or sodium carboxymethylcellulose.

[0043] In the preferred technical solution of the present invention, the buffer is selected from any one of phosphate buffer, citrate buffer, sodium chloride buffer or a combination thereof. Preferably, the buffer is sodium chloride or phosphate buffer.

[0044] In a preferred technical solution of the present invention, the preparation further comprises any one of polyols, amino acids, polypeptides, proteins, nucleic acids, vitamins, polysaccharides, and local anesthetics, or a combination thereof.

[0045] In a preferred technical solution of the present invention, the preparation is selected from any one of a liquid injection or a powder injection.

[0046] In a preferred technical solution of the present invention, the liquid injection is selected from any one of a hydrogel, a suspension, a solution, or a combination thereof.

[0047] Another object of the present invention is to provide a combined use method of an injectable filler preparation, wherein the injectable filler preparation is used in combination with any one of other types of injectable fillers, anesthetics, anti-inflammatory agents, antiallergic agents or a combination thereof; the filler is polycaprolactone microparticles, the polycaprolactone microparticles are spherical particles, the molecular weight of the polycaprolactone is 5000-30000, the particle size of the spherical particles is 20-100 μm, and the polycaprolactone microparticles have a rough surface or a non-smooth surface.

[0048] In the preferred technical solution of the present invention, the other types of injectable fillers are selected from any one of collagen, hyaluronic acid, polymethyl methacrylate, polyacrylamide, silicone, autologous fat or a combination thereof.

[0049] In the preferred technical solution of the present invention, the anesthetic is selected from any one of lidocaine, procaine, tetracaine, bupivacaine, ropivacaine, diclofenac, morphine, hydrocodone, oxycodone, codeine, fentanyl, sodium pentobarbital, sodium phenobarbital, sodium thiopental, chloralose, ethyl carbamate, and chloral hydrate, or a combination thereof.

[0050] In the preferred technical solution of the present invention, the anti-inflammatory agent is selected from any one of steroidal anti-inflammatory agents and non-steroidal anti-inflammatory agents or a combination thereof.

[0051] In the preferred technical solution of the present invention, the steroid anti-inflammatory agent is selected from any one of fluocinolone, hydrocortisone, betamethasone or a combination thereof.

[0052] In the preferred technical scheme of the present invention, the non-steroidal anti-inflammatory agent is selected from any one of aspirin, magnesium salicylate, sodium salicylate, choline magnesium salicylate, diflunisal, disalsalate, ibuprofen, indomethacin, flurbiprofen, phenoxyibuprofen, naproxen, nabumetone, piroxicam, phenylbutazone, diclofenac, fenprofen, ketoprofen, ketorolac, tetraclofenamic acid, sulindac, and tolmetin, or a combination thereof.

[0053] In the preferred technical scheme of the present invention, the antiallergic agent is selected from any one of diphenhydramine, promethazine, chlorpheniramine, sodium cromoglycate, ketotifen, betahistine, montelukast, zafirlukast, salbutamol, calcium gluconate, and adrenal glucocorticoids, or a combination thereof.

[0054] Another object of the present invention is to provide an application of an injectable filler in the preparation of an injectable filler, wherein the filler is polycaprolactone particles, the polycaprolactone particles are spherical particles, the particle size of the spherical particles is 20-100 μm, and the polycaprolactone particles have a rough surface or a non-smooth surface.

[0055] In a preferred technical solution of the present invention, the injection filling site is selected from any one of the face, neck, abdomen, chest, buttocks, thigh, calf, upper arm, and lower arm, or a combination thereof.

[0056] In a preferred technical solution of the present invention, the injectable filler is used to improve any one or a combination of facial emaciation, fat atrophy, sunken cheeks, sunken eye sockets, and skin wrinkles in patients.

[0057] In a preferred technical solution of the present invention, the injectable filler is used in preparing a composition for injectable filling of facial wrinkles.

[0058] In a preferred technical solution of the present invention, the facial wrinkles are selected from any one of shallow to deep nasolabial folds, between the eyebrows, forehead, and corners of the eyes, or a combination thereof.

[0059] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0060] The present invention adopts gel permeation chromatography (GPC) method (Chinese Pharmacopoeia 2020 Edition Part Four General Rules 0514) to detect the molecular weight of polycaprolactone, as shown below:

[0061] Take an appropriate amount of the test sample solution, weigh it accurately, add chloroform to dissolve and dilute it to make a solution containing about 10 mg per 1 mL, filter it with a 0.22 μm organic filter membrane, and take the filtrate.

[0062] For the reference solution, take appropriate amounts of 5-6 polystyrene reference substances of different molecular weights, weigh them accurately, dissolve them in chloroform and dilute them to make a solution containing about 2 mg of each reference substance per 1 mL, dissolve them and mix them evenly.

[0063] The chromatographic conditions were: a gel column (Styragel HR 4E THF, 7.8×300 mm or a column of equivalent performance); the mobile phase was chloroform; the flow rate was 0.7 mL per minute; the detector was a differential refractive index detector; the column temperature was 35° C.; and the injection volume was 10 μL.

[0064] Determination method: Precisely measure the test solution and reference solution, inject them into the liquid chromatograph, and record the chromatogram. The regression equation of the reference solution and the weight average molecular weight (Mw), number average molecular weight (Mn) and molecular weight distribution (Mw / Mn) of the test sample are calculated by GPC software.

[0065] The present invention adopts an electronic tensile testing machine to measure the pushing force: the use speed is 30 mm / min, the stopping force value is 250.00 N, and the stopping displacement is 15.0 mm.

[0066] The present invention uses Kinexus Pro+ rheometer shear viscosity and rheological properties.

[0067] Shear viscosity test: Select rotor model: PU20 L1917 SS, set gap: 0.3mm; after loading the sample, select shear rate scanning program, set temperature to 25℃, and start shear rate to 0.01s -1 , the end shear rate is 100.00s -1 The scanning time is 3 min, and the number of sampling points for each order of magnitude is 20. The shear rate is 1.00 s -1 Shear viscosity at .

[0068] Select the rotor model: PU20 L1917 SS, set the gap: 0.3mm. After loading the sample, select the frequency table (strain control), set the temperature to 25℃, the starting frequency to 10Hz, the ending frequency to 0.1Hz, the shear strain to 0.5%, and the number of sampling points for each order of magnitude to 10, and obtain G' and G" at a frequency of 1Hz.

[0069] Compared with the prior art, the present invention has the following beneficial technical effects:

[0070] 1. The polycaprolactone microspheres in the present invention have a rough surface or a non-smooth surface, which is related to factors such as volatilization efficiency. Compared with a smooth surface, the polycaprolactone microspheres with a rough surface and a porous structure have a higher specific surface area, thereby increasing the contact area with the cells, improving the adhesion ability and retention time of the cells, improving the affinity of the polycaprolactone microspheres, stimulating and accelerating the growth of collagen, and reducing the irritation to the local skin.

[0071] 2. The present invention scientifically screens the dosage of each component during the preparation process, and on the basis of controlling the viscosity of the aqueous solution, cooperates with the rapid membrane emulsification method to obtain regular, rough-surfaced, uniform-size spherical polycaprolactone microparticles.

[0072] 3. The polycaprolactone microspheres obtained by the preparation method of the present invention have a narrow particle size distribution range and uniform particle size, thereby improving the yield of injectable polycaprolactone microspheres.

[0073] 4. The polycaprolactone microspheres prepared in the present invention have uniform particle size distribution and can be quickly dispersed evenly in water for injection, thus avoiding defects such as aggregation, needle hole clogging and nodule formation during injection, and reducing irritation reactions such as skin hemorrhage and edema.

[0074] 5. The preparation method is simple in process, easy to operate, mild in conditions, short in time, and easy to scale up for production. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 Scanning electron microscopy test results of polymer microspheres in Example 1;

[0076] Figure 2 Gross photograph of filler 3 days after subcutaneous injection in rats (D4);

[0077] Figure 3 Gross photograph of filler 2 weeks after subcutaneous injection in rats (D15);

[0078] Figure 4 Gross photograph of filler in rats 8 weeks after subcutaneous injection (D55);

[0079] Figure 5 HE staining 3 days after subcutaneous injection in rats (D4);

[0080] Figure 6 3 days after subcutaneous injection in rats (D4), A: PSR staining; B: α-SMA immunohistochemistry;

[0081] Figure 7 HE staining of rats 2 weeks after subcutaneous injection (D15);

[0082] Figure 8 Two weeks after subcutaneous injection in rats (D15), A: PSR staining; B: α-SMA immunohistochemistry;

[0083] Fig. 9 HE staining of rats 8 weeks after subcutaneous injection (D55);

[0084] Fig.10 8 weeks after subcutaneous injection in rats (D55), A: PSR staining; B: α-SMA immunohistochemistry. DETAILED DESCRIPTION

[0085] The present invention will be specifically described below in conjunction with embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the essence of the present invention.

[0086] Example 1: Preparation of polycaprolactone microspheres

[0087] ① Preparation of aqueous phase: Add 5L of purified water to a 10L reactor, start mechanical stirring, and adjust the rotation power to 25Hz; slowly add 100g of polyvinyl alcohol, heat to 80℃, stir for 5 hours, and cool to room temperature (viscosity 5cP) after the polyvinyl alcohol is completely dissolved;

[0088] ② Preparation of organic phase: Add 500 mL of dichloromethane to a beaker, weigh 100 g of polycaprolactone with a Mn of 20,000 and add it to the beaker, and stir to completely dissolve it;

[0089] ③ Emulsification: adjust the mechanical stirring power to 20 Hz, slowly add the above organic phase into the aqueous phase, stir for 5 min, adjust the pressure to 0.8 bar using a membrane emulsifier, and use a 30 μm membrane tube for rapid membrane emulsification;

[0090] ④ Stirring and volatilizing: transfer the emulsified sample to a 10L reactor, maintain the mechanical stirring power at 20Hz, use nitrogen / compressed air to replace the gas in the reactor, maintain a flow rate of 20m / s, and stir for 24 hours to completely volatilize the organic solvent;

[0091] ⑤ Microsphere collection: After the dichloromethane evaporates, collect the suspension in the reactor, use a centrifuge to separate the solid microspheres (3500rpm, 5min), and wash with purified water and ethanol (wash with purified water 3 times, wash with ethanol once) and centrifuge. Put the sample obtained after centrifugation into a forced air drying oven, adjust the temperature to 40°C, and dry for 20 hours; obtain microspheres with a particle size of 20-80μm.

[0092] Example 2 Preparation of polycaprolactone microspheres

[0093] ① Preparation of aqueous phase: Add 5L of purified water to a 10L reactor, start mechanical stirring, and adjust the rotation power to 25Hz; slowly add 120g of polyvinyl alcohol, heat to 80℃, stir for 6 hours, and cool to room temperature (viscosity 5.8cP) after the polyvinyl alcohol is completely dissolved;

[0094] ② Preparation of organic phase: Add 500 mL of chloroform to a beaker, weigh 110 g of polycaprolactone with a Mn of 22,000 and add it to the beaker, and stir to completely dissolve it;

[0095] ③ Emulsification: Adjust the mechanical stirring power to 25 Hz, slowly add the above organic phase into the aqueous phase, stir for 5 min, adjust the pressure to 1 bar using a membrane emulsifier, and use a 30 μm membrane tube for rapid membrane emulsification;

[0096] ④ Stirring and volatilizing: transfer the emulsified sample to a 10L reactor, maintain the mechanical stirring power at 20Hz, use nitrogen / compressed air to replace the gas in the reactor, maintain a flow rate of 15m / s, and stir for 30 hours to completely volatilize the organic solvent;

[0097] ⑤ Microsphere collection: After the chloroform evaporates, collect the suspension in the reactor, use a centrifuge to separate the solid microspheres (3500rpm, 5min), and wash with purified water and ethanol (wash with purified water 3 times, wash with ethanol once) and centrifuge. Put the sample obtained after centrifugation into a forced air drying oven, adjust the temperature to 40°C, and dry for 20 hours; obtain microspheres with a particle size of 20-80μm.

[0098] Example 3 Preparation of polycaprolactone microspheres

[0099] ① Preparation of aqueous phase: Add 5L of purified water to a 10L reactor, start mechanical stirring, and adjust the rotation power to 25Hz; slowly add 80g of polyvinyl alcohol, heat to 80℃, stir for 4 hours, and cool to room temperature (viscosity 4.7cP) after the polyvinyl alcohol is completely dissolved;

[0100] ② Preparation of organic phase: Add 500 mL of dichloromethane to a beaker, weigh 80 g of polycaprolactone with a Mn of 20,000 and add it to the beaker, and stir to completely dissolve it;

[0101] ③ Emulsification: adjust the mechanical stirring power to 20 Hz, slowly add the above organic phase into the aqueous phase, stir for 5 min, adjust the pressure to 0.8 bar using a membrane emulsifier, and use a 30 μm membrane tube for rapid membrane emulsification;

[0102] ④ Stirring and volatilizing: transfer the emulsified sample to a 10L reactor, maintain the mechanical stirring power at 20Hz, use nitrogen / compressed air to replace the gas in the reactor, maintain a flow rate of 20m / s, and stir for 30 hours to completely volatilize the organic solvent;

[0103] ⑤ Microsphere collection: After the dichloromethane evaporates, collect the suspension in the reactor, filter the microspheres, wash them with purified water and ethanol (wash them 3 times with purified water and once with ethanol), and centrifuge them. Put the sample obtained after centrifugation into a forced air drying oven, adjust the temperature to 40°C, and dry it for 20 hours; obtain microspheres with a particle size of 20-80 μm.

[0104] Example 4 Preparation of polycaprolactone microspheres

[0105] ① Preparation of aqueous phase: Add 5L of purified water to a 10L reactor, start mechanical stirring, and adjust the rotation power to 25Hz; slowly add 60g of methylcellulose, heat to 80℃, stir for 4 hours, and cool to room temperature (viscosity 7cP) after the methylcellulose is completely dissolved;

[0106] ② Preparation of organic phase: Add 500 mL of dichloromethane to a beaker, weigh 100 g of polycaprolactone with a Mn of 18,000 and add it to the beaker, and stir to completely dissolve it;

[0107] ③ Emulsification: adjust the mechanical stirring power to 20 Hz, slowly add the above organic phase into the aqueous phase, stir for 5 min, adjust the pressure to 0.8 bar using a membrane emulsifier, and use a 30 μm membrane tube for rapid membrane emulsification;

[0108] ④ Stirring and volatilizing: Transfer the emulsified sample to a 10L reactor, maintain the mechanical stirring power at 20Hz, use nitrogen / compressed air to replace the gas in the reactor, maintain a flow rate of 20m / s, and stir for 28 hours to completely evaporate the organic solvent.

[0109] ⑤ Microsphere collection: After the dichloromethane evaporates, collect the suspension in the reactor, use a centrifuge to separate the solid microspheres (3500rpm, 5min), and wash with purified water and ethanol (wash with purified water 3 times, wash with ethanol once) and centrifuge. Put the sample obtained after centrifugation into a forced air drying oven, adjust the temperature to 40°C, and dry for 20 hours; obtain microspheres with a particle size of 20-80μm.

[0110] Example 5 Preparation of polycaprolactone preparation

[0111] Weigh 3 g of sodium carboxymethyl cellulose (model 3000-5000 Pa·s), add 96 g of phosphate solution and 1 g of glycerol, stir to dissolve, add 49 g of PCL microspheres, mix evenly to obtain the product. The method of the present invention is used to test the pushing force, viscoelasticity and shear viscosity.

[0112] Average pushing force: 22.543N (27G*12MM needle speed 30mm / min)

[0113] Shear viscosity: 310.6Pa·s

[0114] Viscoelasticity: G': 456.8Pa G': 547.6Pa at 1Hz

[0115] Example 6 Preparation of polycaprolactone preparation

[0116] Weigh 3 g of sodium carboxymethyl cellulose (model 9000-11000 Pa·s), add 96 g of phosphate solution and 1 g of glycerol, stir to dissolve, add 49 g of PCL microspheres, mix well and obtain. Test the extrusion force, viscoelasticity and shear viscosity.

[0117] Average pushing force: 24.448N (27G*12MM needle speed 30mm / min)

[0118] Shear viscosity: 295.3Pa·s

[0119] Viscoelasticity: G': 844.8Pa G”: 769.4Pa

[0120] Example 7 Preparation of polycaprolactone preparation

[0121] Weigh 3 g of sodium carboxymethyl cellulose (model 12000-15000 Pa·s), add 96 g of phosphate solution and 1 g of glycerol, stir to dissolve, add 49 g of PCL microspheres, mix well and obtain. Test the extrusion force, viscoelasticity and shear viscosity.

[0122] Average pushing force: 28.005N (27G*12MM needle speed 30mm / min)

[0123] Shear viscosity: 277.4Pa·s

[0124] Viscoelasticity: 1Hz G': 1097Pa G”: 942.6Pa

[0125] Example 8 Preparation of polycaprolactone preparation

[0126] Weigh 90 g of cross-linked sodium hyaluronate gel (20 mg / g) and 10 g of non-cross-linked sodium hyaluronate solution (20 mg / g), stir and mix them evenly, then add 49 g of PCL microspheres and mix them evenly to obtain the product. Test the extrusion force, viscoelasticity, and shear viscosity.

[0127] Average pushing force: 28.005N (27G*12MM needle speed 30mm / min)

[0128] Shear viscosity: 204.2Pa·s

[0129] Viscoelasticity: 1Hz G'844.1Pa G"102.9Pa

[0130] Example 9 Preparation of polycaprolactone preparation

[0131] Weigh 72 g of cross-linked sodium hyaluronate gel (20 mg / g), 8 g of non-cross-linked sodium hyaluronate solution (20 mg / g), and 20 g of phosphate solution, stir to dissolve, add 49 g of PCL microspheres, and mix well to obtain the product. Test the extrusion force, viscoelasticity, and shear viscosity.

[0132] Average pushing force: 24.071N (27G*12MM needle speed 30mm / min)

[0133] Shear viscosity: 160.7Pa·s

[0134] Viscoelasticity: G': 263.2Pa G': 56.61Pa at 1Hz

[0135] Example 10 Preparation of polycaprolactone preparation

[0136] Weigh 54 g of cross-linked sodium hyaluronate gel (20 mg / g), 6 g of non-cross-linked sodium hyaluronate solution (20 mg / g), and 40 g of phosphate solution, stir to dissolve, add 49 g of PCL microspheres, and mix well to obtain the product. Test the extrusion force, viscoelasticity, and shear viscosity.

[0137] Average pushing force: 16.564N (27G 1*12MM needle speed 30mm / min)

[0138] Shear viscosity: 116.5Pa·s

[0139] Viscoelasticity: G': 187.1Pa G': 33.67Pa at 1Hz

[0140] Test Example 1 Local reaction test of the polycaprolactone product of the present invention and the commercially available Ellanse (girl needle) product on SD rats

[0141] (1) Experimental animals

[0142] Animals: 120 Sprague-Dawley (SD) rats, female;

[0143] Age: about 6-8 weeks, weight: 200-220g;

[0144] Animal grade: SPF grade;

[0145] Source of experimental animals: Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0146] Rats were randomly divided into 4 groups: blank control group, PCL-1 group (Example 5), PCL-2 group (commercially available Ellanse-S), and PCL-3 group (commercially available Ellanse-M), and local reaction tests were performed on the injection. The injection filling was performed on the back of the rats, and the injection volume was 100 mg / kg; the blank control group was injected with the same amount of normal saline.

[0147] (2) General condition of animals

[0148] During the experiment, no animals in the blank control group and all experimental groups died, and the appearance, signs, behavior, activities, food intake, water drinking, defecation, etc. of all animals were normal.

[0149] (3) Injection site conditions

[0150] Immediately after injection, the skin of the animals in the control group showed obvious bulges at the injection site, which returned to flatness within 30 minutes after injection and did not reappear during the subsequent observation period.

[0151] Immediately after injection, the skin of all animals in the three PCL experimental groups was significantly raised at the injection site; 24 hours after injection, the area of ​​local bulge in the injection site expanded and the amplitude of bulge decreased; during the subsequent observation period, most animals could feel the presence of subcutaneous foreign bodies when observed with the naked eye or palpated. There was no significant difference in the above changes among the three products.

[0152] Immediately to 8 hours after injection, 12 / 30, 18 / 30, and 17 / 30 animals in the PCL-1, PCL-2, and PCL-3 groups, respectively, had local skin redness, which all recovered completely within 3 days after injection.

[0153] (4) Routine observation

[0154] The filler was visible under the skin at the injection site of all dissected animals in the three PCL experimental groups, and no obvious migration was observed relative to the puncture site. Three days after injection, no abnormality was observed in the connective tissue around the injection site in the control group; the subcutaneous connective tissue around the filler in the animals of the three PCL product groups was thickened (edema) to varying degrees, among which the degree / range of thickening of the connective tissue around the filler was significantly greater in 3 / 5 and 4 / 5 animals in the PCL-2 and PCL-3 groups, respectively.

[0155] (5) Filling status

[0156] Color: 3 days (D4), 2 weeks (D15), and 8 weeks (D55) after injection, all the fillers obtained from the three PCL experimental groups were white / milky white.

[0157] Shape: 3 days after injection (D4, Figure 1), all the fillers in the three PCL experimental groups were soft gel-like, with an irregular shape. The shape of the fillers changed greatly with the stretching of the skin; 2 weeks after injection (D15, Figure 2 ) and 8 weeks (D55, Figure 3 ), all the fillers were relatively fixed in shape, irregular oval / long strip, with smooth outer edges, and the fillers were wrapped by fascia with clear boundaries with subcutaneous tissue.

[0158] Size: 3 days after injection (D4), the surface area measurement was not statistically compared because the filler had not yet been fixed. 2 weeks after injection (D15), no significant difference was found in the surface area of ​​the fillers in the three PCL experimental groups. 8 weeks after injection (D55), no significant difference was found in the surface area of ​​the fillers in the three PCL experimental groups.

[0159] (6) Histopathology

[0160] 3 days after injection (D4, Figure 4-5 ), the connective tissue around the three PCL fillers was thickened, showing acute inflammatory exudation, and neutrophils and cellulose were seen in the exudate. The fillers were wrapped by connective tissue, and a large number of leukocytes, mainly neutrophils, were seen infiltrating between the PCL particles in the connective tissue and the superficial area (periphery) of the fillers. However, no new tissue / cell infiltration was seen in the deep layer (inside) of the fillers, and they fell off during the film making process. A large amount of unformed extracellular matrix and a small amount of fibrous tissue were seen between the PCL particles in the superficial area (periphery) of the fillers. The fibrous tissue was mainly composed of type I and type III collagen, and the proportion of unstable type III collagen was high; myofibroblasts and new capillaries were seen in the interstitium.

[0161] 2 weeks after injection (D15, Figure 6-7 ), the connective tissue around the three PCL fillers did not thicken, and no inflammatory exudate was observed. Typical granulomatous inflammatory cell infiltration and new tissue formation were observed between PCL particles in the superficial area (periphery) of the filler. Compared with 3 days after injection (D4), the acute inflammation around the filler subsided, and a foreign body granulomatous inflammatory reaction dominated by macrophages was observed in the superficial area (periphery) of the filler, and the new tissue further invaded the deep layer of the filler; a large number of macrophages and their derived epithelial cells and tissue cells were seen between PCL particles, and a small number of multinucleated giant cells were seen; extracellular matrix and fibrous tissue dominated by type I and type III collagen fibers were seen, and the proportion of type I collagen fibers increased; a large number of myofibroblasts and new capillaries were seen in the interstitium. At this time point, the local tissue reactions caused by the three PCL products were roughly equivalent.

[0162] 8 weeks after injection (D55, Figure 8-9), the local tissue reactions caused by the three PCL fillers were still mainly granulomatous inflammatory reactions and the formation of new tissues. Compared with the injection for 4 weeks (D29), the granulomatous inflammatory reactions caused by the three PCL fillers tended to be stable, and the proportion of epithelial cells and tissue cells decreased; the proportion of multinucleated giant cells did not change significantly in the PCL-1 group (Example 5) and the PCL-3 group (Ellanse-M), but increased significantly in the PCL-2 group (Ellanse-S); the PCL-1 group (Example 5) had less inflammatory exudate compared with the PCL-2 group (Ellanse-S) and the PCL-3 group (Ellanse-M). The new tissues caused by the three PCL fillers continued to grow and mature, showing interstitial expansion, increased / thickened extracellular matrix and fibrous tissue, and formed a complete fiber network with clear boundaries and regular shapes. The proportion of more stable type I collagen fibers increased significantly; the number of myofibroblasts decreased.

[0163] The above description is only an example of the implementation mode of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an injection filler, characterized in that: The steps include: (1) Preparation of aqueous solution: adding a surfactant to purified water, stirring and dissolving; the viscosity of the aqueous solution is 2-10 cP; (2) Preparation of organic phase solution: adding polycaprolactone into an organic solvent and stirring until completely dissolved; (3) Emulsification granulation: the organic phase solution and the aqueous phase solution are mixed uniformly, stirred, and emulsified using a membrane emulsifier to prepare microspheres; (4) Stirring and volatilizing: Stirring to evaporate all the organic solvent; (5) Microsphere collection: Collect the suspension, filter, wash, and dry to obtain injectable filler microspheres.

2. The preparation method according to claim 1, characterized in that: The preparation method satisfies at least one of the following conditions (A) to (S): (A) In step (1), the surfactant is any one of polyvinyl alcohol, methyl cellulose, polyoxyethylene, polyoxypropylene, polyethyleneimine, polystyrene, polyoxysilane, and hydroxypropyl cellulose, or a combination thereof; (B) In step (1), the ratio of the surfactant to the purified water is 10-50 g / L, preferably 15-40 g / L, more preferably 20-30 g / L; (C) In step (1), the viscosity of the aqueous solution is 4-8 cP; (D) In ​​step (1), the stirring method is mechanical stirring, and the rotation power of the mechanical stirrer is 10-50 Hz, preferably 20-30 Hz; (E) In step (1), during the stirring, the temperature of the system is 60-120° C., and the stirring is performed for 2-6 hours, and the temperature is cooled to room temperature after the surfactant is completely dissolved; (F) In step (2), the organic solvent is any one of acetone, ethanol, ethyl acetate, chloroform, toluene and dichloromethane, or a combination thereof; (G) In step (2), the ratio of the polycaprolactone to the organic solvent is 100-300 g / L, preferably 150-250 g / L; (H) In step (2), the molecular weight of the polycaprolactone is 5000-30000, preferably 10000-25000, more preferably 15000-22000; (I) In step (3), the stirring method is mechanical stirring, and the rotation power of the mechanical stirrer is 10-50 Hz; (J) In step (3), the stirring time is 3-10 min; (K) In step (3), the emulsification pressure of the membrane emulsifier is 0.5-1 bar; (L) In step (3), the pore size of the membrane tube of the membrane emulsifier is 30 μm; (M) In step (4), the stirring method is mechanical stirring, and the rotation power of the mechanical stirrer is 10-50 Hz; (N) In step (4), the gas in the reaction kettle is extracted using a vacuum pump, the vacuum degree is maintained at -0.04 to -0.1 MPa, and the reaction mixture is stirred for 12 to 24 hours to completely evaporate the organic solvent; (O) In step (5), the filtering operation is replaced by a centrifugation and / or sedimentation operation; preferably, when the filtering operation is replaced by the centrifugation operation, the centrifugation speed is 2000-5000 rpm, more preferably 3000-4000 rpm; preferably, when the filtering operation is replaced by the centrifugation operation, the centrifugation time is 2-8 min, more preferably 3-6 min; (P) During the cleaning process of step (5), the purified water and ethanol are used for cleaning; preferably, during the cleaning process, the purified water is used for cleaning 2-5 times, and the ethanol is used for cleaning 1-2 times; (Q) In step (5), the washing step further comprises centrifugation to collect the microspheres; (R) In step (5), the drying temperature is 30 to 60°C, preferably 30 to 40°C; (S) In step (5), the drying time is 10 to 30 hours, preferably 15 to 24 hours.

3. An injection filler, characterized in that: The injection filler is prepared by the preparation method of the injection filler as claimed in claim 1 or 2.

4. The injection filler according to claim 3, characterized in that: The injectable filler is polycaprolactone microparticles, the particle size of the polycaprolactone microspheres is 20-100 μm, and has a rough surface or a non-smooth surface; Preferably, the rough surface or non-smooth surface of the polycaprolactone particles has irregular pore sizes; Preferably, the particle size of the polycaprolactone microspheres is 20-70 μm, more preferably 25-50 μm.

5. An injectable filler preparation, characterized in that: Comprising the injection filler as claimed in claim 3 or 4; Preferably, the preparation further comprises any one or a combination of a suspension stabilizer and a buffer.

6. The injection filler preparation according to claim 5, characterized in that The injectable filler preparation satisfies at least one of the following conditions (1) to (4): (1) The suspension stabilizer is selected from any one or a combination of polyvinyl alcohol (PVA), gelatin, gum arabic, guar gum, chondroitin sulfate, hyaluronic acid (HA), cross-linked hyaluronic acid, sodium carboxymethyl cellulose (CMC), polyvinyl pyrrolidone (PVP), methyl cellulose (MC), hydroxypropyl methyl cellulose (HPMC), starch, pectin acid, heparin, glucose, β-cyclodextrin, chitosan, sodium alginate; preferably, the suspension stabilizer is any one or a combination of hyaluronic acid, cross-linked hyaluronic acid, sodium carboxymethyl cellulose; (2) The buffer is selected from any one of phosphate buffer, citrate buffer, sodium chloride buffer or a combination thereof; preferably, the buffer is sodium chloride or phosphate buffer; (3) The preparation further comprises any one or a combination of polyols, amino acids, polypeptides, proteins, nucleic acids, vitamins, polysaccharides, and local anesthetics; (4) The preparation is selected from any one of a liquid injection or a powder injection; preferably, the liquid injection is selected from any one of a hydrogel, a suspension, a solution or a combination thereof.

7. A combined use of the injection filler preparation according to claim 5 or 6, characterized in that: The injectable filler preparation is used in combination with any one of other types of injectable fillers, anesthetics, anti-inflammatory agents, anti-allergic agents, or a combination thereof.

8. The combined use of the injection filler preparation according to claim 7, characterized in that: The combination of the injection filler preparation satisfies at least one of the following conditions (1) to (4): (1) The other types of injectable fillers are selected from any one or a combination of collagen, hyaluronic acid, polymethyl methacrylate, polyacrylamide, silicone, and autologous fat; (2) the anesthetic is selected from any one of lidocaine, procaine, tetracaine, bupivacaine, ropivacaine, diclofenac, morphine, hydrocodone, oxycodone, codeine, fentanyl, sodium pentobarbital, sodium phenobarbital, sodium thiopental, chloralose, ethyl carbamate, and chloral hydrate, or a combination thereof; (3) The anti-inflammatory agent is selected from any one or a combination of steroidal anti-inflammatory agents and non-steroidal anti-inflammatory agents; preferably, the steroidal anti-inflammatory agent is selected from any one or a combination of fluocinolone, hydrocortisone, and betamethasone; preferably, the non-steroidal anti-inflammatory agent is selected from any one or a combination of aspirin, magnesium salicylate, sodium salicylate, choline magnesium salicylate, diflunisal, disalsalate, ibuprofen, indomethacin, flurbiprofen, phenoxyibuprofen, naproxen, nabumetone, piroxicam, phenylbutazone, diclofenac, fenprofen, ketoprofen, ketorolac, tetraclofenamic acid, sulindac, and tolmetin; (4) The antiallergic agent is selected from any one of diphenhydramine, promethazine, chlorpheniramine, sodium cromoglycate, ketotifen, betahistine, montelukast, zafirlukast, salbutamol, calcium gluconate, and adrenal glucocorticoids, or a combination thereof.

9. Use of the injection filler prepared by the preparation method according to claim 1 or 2 as an injection filler; Preferably, the filling site of the injectable filler is selected from any one of the face, neck, abdomen, chest, buttocks, thighs, calves, upper arms, and lower arms, or a combination thereof, and more preferably the filling site is the face.

10. Use of the injectable filler preparation according to claim 5 or 6 for preparing a composition for injectable filling of facial wrinkles.

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